Polymer for detection of target substance, and method for detection of target substance

Abstract

Provided is a method of detecting a target substance by which the target substance can be detected with efficiency and a high sensitivity, and in a simple manner, a target substance detection polymer used in the method, and a method of forming the polymer. The method of detecting a target substance includes the steps of: (A) forming a target substance detection polymer by causing multiple kinds of nucleic acid probes for forming a polymer to react with a binding probe having a region capable of binding to the target substance and a region capable of binding to at least one of the nucleic acid probes in a solution; (B) binding the target substance detection polymer and the target substance; and (C) detecting the target substance detection polymer to which the target substance is bound.

1 . A method of detecting a target substance, comprising the steps of:
 (A) forming a target substance detection polymer by causing multiple kinds of nucleic acid probes for forming a polymer to react with a binding probe comprising a target region capable of directly or indirectly binding to the target substance and a region capable of binding to at least one of the nucleic acid probes in a solution;   (B) binding the target substance to the target substance detection polymer; and   (C) detecting the target substance detection polymer to which the target substance is bound,   wherein the multiple kinds of nucleic acid probes comprise   a nucleic acid probe comprising at least n (n≧3) nucleic acid regions formed of a nucleic acid region X 1 ′ a nucleic acid region X 2 , . . . , and a nucleic acid region X n  from a 5′ terminal side in the stated order, and   a nucleic acid probe comprising at least n (n≧3) nucleic acid regions formed of a nucleic acid region X 1 ′ complementary to the nucleic acid region X 1 , a nucleic acid region X 2 ′ complementary to the nucleic acid region X 2 , . . . , and a nucleic acid region X n ′ complementary to the nucleic acid region X n  from a 5′ terminal side in the stated order.   
     
     
         2 . A method of detecting a target substance, comprising the steps of:
 (A) forming a target substance detection polymer by causing multiple kinds of nucleic acid probes for forming a polymer to react with a binding probe comprising a target region capable of directly or indirectly binding to the target substance and a region capable of binding to at least one of the nucleic acid probes in a solution;   (B) binding the target substance to the target substance detection polymer; and   (C) detecting the target substance detection polymer to which the target substance is bound,   wherein the multiple kinds of nucleic acid probes each comprise a base sequence that hybridizes with any other kind of nucleic acid probe as represented by the following formula (I):   
       
         
           
           
               
               
           
         
       
       wherein two straight lines illustrated in a ladder fashion represent nucleic acids that hybridize with each other. 
     
     
         3 . A method of detecting a target substance, comprising the steps of:
 (A) forming a target substance detection polymer by causing multiple kinds of nucleic acid probes for forming a polymer to react with a binding probe comprising a target region capable of directly or indirectly binding to the target substance and a region capable of binding to at least one of the nucleic acid probes in a solution;   (B) binding the target substance to the target substance detection polymer; and   (C) detecting the target substance detection polymer to which the target substance is bound,   wherein the multiple kinds of nucleic acid probes each comprise a base sequence that hybridizes with any other kind of nucleic acid probe as represented by the following formula (II):   
       
         
           
           
               
               
           
         
       
       wherein two straight lines illustrated in a ladder fashion represent nucleic acids that hybridize with each other. 
     
     
         4 . A method of detecting a target substance according to  claim 1 , wherein the formation step comprises
 a first hybridization step of causing the multiple kinds of nucleic acid probes to hybridize with each other to form a first polymer and   a second hybridization step of causing the first polymer and the binding probe to hybridize with each other to form the target substance detection polymer.   
     
     
         5 . A method of detecting a target substance according to  claim 1 , wherein the formation step comprises the step of causing the multiple kinds of nucleic acid probes and the binding probe to hybridize with one another simultaneously. 
     
     
         6 . A method of detecting a target substance according to  claim 1 , further comprising the step of diluting a solution containing the target substance detection polymer after the formation step to prepare a detection solution. 
     
     
         7 . A method of detecting a target substance according to  claim 1 , wherein at least one kind of nucleic acid probe of the multiple kinds of nucleic acid probes is labeled with a labeling substance. 
     
     
         8 . A method of detecting a target substance according to  claim 1 , wherein the target region of the binding probe comprises a portion capable of specifically binding to the target substance. 
     
     
         9 . A method of detecting a target substance according to  claim 8 , wherein:
 the target substance comprises a nucleic acid; and   the target region of the binding probe comprises a nucleic acid comprising a base sequence complementary to that of the target nucleic acid.   
     
     
         10 . A method of detecting a target substance according to  claim 1 , wherein the target substance detection polymer and the target substance are bound to each other through a spacer substance comprising a region capable of binding to the target substance and a region capable of binding to the target region of the binding probe. 
     
     
         11 . A method of detecting a target substance according to  claim 1 , wherein the target substance detection polymer is bound to avidin or biotin through the binding probe. 
     
     
         12 . A method of detecting a target substance according to  claim 11 , wherein the binding step comprises the step of binding the target substance to the target substance detection polymer through a bond between biotin and avidin. 
     
     
         13 . A method of forming a target substance detection polymer, the method comprising the step of forming a target substance detection polymer by causing multiple kinds of nucleic acid probes for forming a polymer to hybridize with a binding probe comprising a target region capable of directly or indirectly binding to the target substance and a region capable of binding to at least one of the nucleic acid probes,
 wherein:   the binding probe is free of being bound to the target substance; and   the multiple kinds of nucleic acid probes comprise   a nucleic acid probe comprising at least n (n≧3) nucleic acid regions formed of a nucleic acid region X 1 , a nucleic acid region X 2 , . . . , and a nucleic acid region X n  from a 5′ terminal side in the stated order, and   a nucleic acid probe comprising at least n (n≧3) nucleic acid regions formed of a nucleic acid region X 1 ′ complementary to the nucleic acid region X 1 , a nucleic acid region X 2 ′ complementary to the nucleic acid region X 2 , . . . , and a nucleic acid region X n ′ complementary to the nucleic acid region X n  from a 5′ terminal side in the stated order.   
     
     
         14 . A method of forming a target substance detection polymer, the method comprising the step of forming the target substance detection polymer by causing multiple kinds of nucleic acid probes for forming a polymer to hybridize with a binding probe comprising a target region capable of directly or indirectly binding to the target substance and a region capable of binding to at least one of the nucleic acid probes,
 wherein:   the binding probe is free of being bound to the target substance; and   the multiple kinds of nucleic acid probes each comprise a base sequence that hybridizes with any other kind of nucleic acid probe as represented by the following formula (I):   
       
         
           
           
               
               
           
         
       
       wherein two straight lines illustrated in a ladder fashion represent nucleic acids that hybridize with each other. 
     
     
         15 . A method of forming a target substance detection polymer, the method comprising the step of forming the target substance detection polymer by causing multiple kinds of nucleic acid probes for forming a polymer to hybridize with a binding probe comprising a target region capable of directly or indirectly binding to the target substance and a region capable of binding to at least one of the nucleic acid probes,
 wherein:   the binding probe is free of being bound to the target substance; and   the multiple kinds of nucleic acid probes each comprise a base sequence that hybridizes with any other kind of nucleic acid-probe as represented by the following formula (II):   
       
         
           
           
               
               
           
         
       
       wherein two straight lines illustrated in a ladder fashion represent nucleic acids that hybridize with each other. 
     
     
         16 . A method of forming a target substance detection polymer according to  claim 13 , wherein the formation step comprises
 a first hybridization step of causing the multiple kinds of nucleic acid probes to hybridize with each other to form a first polymer and   a second hybridization step of causing the first polymer and the binding probe to hybridize with each other to form the target substance detection polymer.   
     
     
         17 . A method of forming a target substance detection polymer according to  claim 13 , wherein the formation step comprises the step of causing the multiple kinds of nucleic acid probes and the binding probe to hybridize with one another simultaneously. 
     
     
         18 . A method of forming a target substance detection polymer according to  claim 13 , wherein at least one kind of nucleic acid probe of the multiple kinds of nucleic acid probes is labeled with a labeling substance. 
     
     
         19 . A method of forming a target substance detection polymer according to  claim 13 , wherein the target region of the binding probe comprises a portion capable of specifically binding to the target substance. 
     
     
         20 . A method of forming a target substance detection polymer according to  claim 19 , wherein:
 the target substance comprises a nucleic acid; and   the target region of the binding probe comprises a nucleic acid comprising a base sequence complementary to that of the target nucleic acid.   
     
     
         21 . A method of forming a target substance detection polymer according to  claim 13 , wherein the target substance detection polymer is bound to avidin or biotin through the binding probe. 
     
     
         22 . A method of forming a target substance detection polymer according to  claim 21 , wherein the target region of the binding probe is bound to avidin or biotin. 
     
     
         23 . A method of forming a target substance detection polymer according to  claim 21 , wherein:
 the target region of the binding probe comprises a region which is capable of directly or indirectly binding avidin or biotin; and   the formation step comprises the steps of
 forming a second polymer by causing the multiple kinds of nucleic acid probes for forming a polymer to hybridize with the binding probe and 
 binding avidin or biotin to the binding probe of the second polymer. 
   
     
     
         24 . A target substance detection polymer, comprising:
 multiple kinds of nucleic acid probes for forming a polymer; and   a binding probe comprising a target region capable of directly or indirectly binding to the target substance and a region capable of binding to at least one of the nucleic acid probes,   the target substance detection polymer being formed by causing the multiple kinds of nucleic acid probes to hybridize with the binding probe and the binding probe being free of being bound to the target substance,   wherein the multiple kinds of nucleic acid probes comprise   a nucleic acid probe comprising at least n (n≧3) nucleic acid regions formed of a nucleic acid region X 1 , a nucleic acid region X 2 , . . . , and a nucleic acid region X n  from a 5′ terminal side in the stated order, and   a nucleic acid probe comprising at least n (n≧3) nucleic acid regions formed of a nucleic acid region X 1 ′ complementary to the nucleic acid region X 1 , a nucleic acid region X 2 ′ complementary to the nucleic acid region X 2 , . . . , and a nucleic acid region X n ′ complementary to the nucleic acid region X n  from a 5′ terminal side in the stated order.   
     
     
         25 . A target substance detection polymer, comprising:
 multiple kinds of nucleic acid probes for forming a polymer; and   a binding probe comprising a target region capable of directly or indirectly binding to the target substance and a region capable of binding to at least one of the nucleic acid probes,   the target substance detection polymer being formed by causing the multiple kinds of nucleic acid probes to hybridize with the binding probe and the binding probe being free of being bound to the target substance,   wherein the multiple kinds of nucleic acid probes each comprise a base sequence that hybridizes with any other kind of nucleic acid probe as represented by the following formula (I):   
       
         
           
           
               
               
           
         
       
       wherein two straight lines illustrated in a ladder fashion represent nucleic acids that hybridize with each other. 
     
     
         26 . A target substance detection polymer, comprising:
 multiple kinds of nucleic acid probes for forming a polymer; and   a binding probe comprising a target region capable of directly or indirectly binding to the target substance and a region capable of binding to at least one of the nucleic acid probes,   the target substance detection polymer being formed by causing the multiple kinds of nucleic acid probes to hybridize with the binding probe and the binding probe being free of being bound to the target substance,   wherein the multiple kinds of nucleic acid probes each comprise a base sequence that hybridizes with any other kind of nucleic acid probe as represented by the following formula (II):   
       
         
           
           
               
               
           
         
       
       wherein two straight lines illustrated in a ladder fashion represent nucleic acids that hybridize with each other. 
     
     
         27 . A target substance detection polymer according to  claim 24 , wherein the target region of the binding probe comprises a portion capable of specifically binding to the target substance. 
     
     
         28 . A target substance detection polymer according to  claim 27 , wherein:
 the target substance comprises a nucleic acid; and   the target region of the binding probe comprises a nucleic acid comprising a base sequence complementary to that of the target nucleic acid.   
     
     
         29 . A target substance detection polymer according to  claim 24 , wherein the target substance detection polymer is bound to avidin or biotin through the binding probe. 
     
     
         30 . A target substance detection polymer formed by the method according to  claim 13 . 
     
     
         31 . A method of detecting a target substance, comprising the steps of:
 binding the target substance to the target substance detection polymer according to  claim 24 ; and   detecting the target substance detection polymer to which the target substance is bound.   
     
     
         32 . A method of detecting a target substance according to  claim 31 , wherein the target substance detection polymer and the target substance are bound to each other through a spacer substance comprising a region capable of binding to the target substance and a region capable of binding to the target region of the binding probe. 
     
     
         33 . A method of detecting a target substance according to  claim 31 , wherein the binding step comprises the step of binding the target substance to the target substance detection polymer according to  claim 29  through a bond between biotin and avidin.